The present invention relates to methods for separating and quantifying urinary porphyrins, and more particularly relates to biological testing methods for separating and then determining the amount of coproporphyrin and uroporphyrin in human urine utilizing chemical analytical techniques involving anionic ion exchange.
Porphyrins are a group of organic pigments normally found as free compounds in minute quantities in the body. Porphyrins have been found to be of major importance to human metabolism. Porphyrins are not normally excreted in any significant amounts in the urine, and those disorders which lead to excretion of porphyrins are referred to as "porphyrinuria." Disorders of porphyrin metabolism are termed "porphyria".
Porphyrins detectable in urine have been divided into two primary fractions denoted as "coproporhyrins" and "uroporphyrins". Those porphyrins designated as "coproporphyrins" are extracted from urine treated with sodium carbonate and aqueous acetic acid and then extracted with ethyl acetate as taught by Schwartz et al "An improved method for the determination of urinary coprophyrin and evaluation of the factors influencing the analysis" J. LAB. CLIN. MED. 37, 843-859 (1951). The remaining porphyrins have been designated as "uroporphyrins" and are extracted from the aqueous phase by adsorption on aluminum oxide followed by dilution with 1.5 N HCl. Uroporphyrins are those porphyrins which are soluble in water but insoluble in ethyl acetate at a pH level of about 4 to 6.
The body contains large amounts of porphyrins as metal complexes which are associated with proteins e.g. hemoglobin, myoglobin, catalase, and cytochromes. Normally, only minimum amounts of free porphyrins are present in the body. However, the amounts of porphyrins found in the urine increase as a result of various malfunctions or problems such as vitamin deficiency, liver damage, (jaundice, infectious hepatitis, cirrosis), lead poisoning, and certain drugs (arsenic, carbon tetrachloride, benzine, and sulfomamides), or as problems in porphyrin metabolism such as congenital erythropoietic porphyria.
While in high concentrations the porphyrins are red to pink, they rarely appear in high enough concentrations in urine so that their color can be noticed. However, minute quantities fluoresce under ultraviolet light and are thus detectable.
Increased COPRO levels in urine are indicative of congenital erythropoietic porphyria and hepatic porphyria, associated with heavy metal poisoning, infectious hepatitis, cirrhosis, and various forms of anemia. Increased URO levels in urine are indicative of congenital erythropoietic porphyria, acute intermittent porphyria, and porphyria cutanea tarda.
Certain techniques and methodologies have been developed for qualitative detection of excreted porphyrins (porphyrinuria). For instance, ion exchange techniques have been utilized which typically require adsorption of a urine sample on an anionic ion exchange resin, elution of substantially all porphyrins with hydrochloric acid, and the determination of the presence or absence of free porphyrins by examining the eluate under long wavelength ultraviolet light whereby the presence of porphyrins is signaled by a pink fluorescence. However, this procedure indicates the presence of either or both COPRO and URO and does not provide for quantitative measurement of all the porphyrins or for the isolation and quantitation of either or both COPRO and URO.
Other quantitative methods of determination of COPRO and URO have been utilized. Perhaps the most widely used is that of Talman et al, "Porphyrins in Urine" Stand. Methods Clin. Chem. 2; 137 (1958). However, this method has not been widely adopted in clinical laboratories because it is multi-step, includes complex procedures, such as solvent extractions, and requires an extensive amount of time to complete each porphyrin determination. More recently Martinez et al "Spectrofluorometric Determination of Porphyrins in Urine" Clin. Chem. 17; 199 (1971) reported fluorometric methods of porphyrin analysis using ion exchange resins. However, these techniques do not include the separation of and selective quantitation of COPRO and URO. Sobel et al, "Separation and Quantitation of Copro Porphyrin and Uro Porphyrin in Urine" Clin. Chem. 20; 1397 (1974) discloses the separation and measurement of COPRO and URO in a system requiring concommitant ion exchange resins and solvent extractions. This latter technique has been questioned in terms of whether the eluates produce background fluoresence interference. Subsequently, Lavallee et al, "Compensation by Zinc Chelation for Fluorescent Background in Determining Copro Porphyrin and Uro Porphyrin in Urine" Clin. Chem. 23; 282 (1977) suggested the elimination of interference in the Sobel et al technique by adding zinc, in the form of ZnCl.sub.2, to urine samples.